Currently the separation of chiral molecules is typically achieved using a chiral stationary phase (CSP) and/or with chiral eluent-selector additive in a mobile phase. Disadvantages of these approaches are expense, narrowness of applicability for a particular CSP or chiral eluent and time consuming development. Previous work (M. Alyamani, et. al <i>J. Steroid Biochem. Mol. Biol.</i> 2016 doi: 10.1016/j.jsbmb.2016.04.002) noted that optimum separation of abiraterone metabolite enantiomer pairs on a C18 reversed-phase column was achieved by an isocratic aqueous mobile phase with acetonitrile/methanol (ACN/MeOH) mixture for an organic modifier. This chiral separation of enantiomer pairs could not be achieved by using only a methanol or only an acetonitrile organic modifier in the mobile phase.<br/>Further investigation was done in the present work to more thoroughly characterize separation of chiral steroid compounds by reversed-phase HPLC.  In the present work the ratio of the ACN/MeOH organic modifier was varied to determine the particular ratio for the optimized separation of a specific pair of enantiomers. In order to have a valid comparison of the different ratios to find the optimum ratio of ACN/MeOH for peak separation, the retention time of the earliest eluting peak of a particular enantiomer pair had to be the same for each tested ACN/MeOH ratio, which was accomplished by varying the water percentage in the mobile phase. The abiraterone metabolite enantiomer pairs (3-keto-5β- abiraterone and 3-keto-5α- abiraterone; 3β-OH-5β- abiraterone and 3α-OH-5β- abiraterone; 3β-OH-5α- abiraterone and 3α-OH-5α-abiraterone) showed a parabolic plot for retention time difference versus the ACN/MeOH with a maximum at a particular ACN/MeOH ratio. However, the 17α-estradiol and 17β-estradiol enantiomer pair showed the best separation of the enantiomers with only an ACN modifier. Properties of the organic modifiers in relation to properties of the enantiomers as possible effectors of the chromatography are discussed.<br/>

Separating chiral steroid compounds by isocratic C18 reversed phase HPLC: Optimization of the acetonitrile/methanol ratio to maximize separation

2019 Fall National Meeting

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